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Trabajo presentado en el 19th International Congress of Developmental Biology, celebrado en Guia (Portugal) del 16 al 20 de octubre de 2022., The animal body plan formation takes place during the embryonic development and it results from the precise spatio-temporal regulation of the gene expression. The animal form evolves largely by altering the expression of functionally conserved developmental genes and the vast networks they control. Most of the genomic signatures underlying key evolutionary processes can be found in the cis-regulatory elements (CREs) that control when, where and how much the genes are transcribed. We have identified genome-wide CREs acting during early development in zebrafish at different embryonic stages. To do that, we performed both ATAC-seq and RNA-seq, which allowed us to link the identified CREs to their target developmental genes and determine how their activity change in the different developmental stages. Several works over the last decades have shown that regulatory DNA can act at long distances, often more than 1 Mb, allowing the contact with promoters of target genes through chromatin loops. These enhancer-promoter physical interactions occur in a highly regulated manner, showing the importance of a precise regulation of the gene expression along development and animal evolution. To identify activated or repressed enhancers upon modulation of the genetic pathways, embryos have been treated with different drugs during the gastrulation period, when body plan basic layout is generated. At this point the open chromatin profiling have been done at three different stages: 80% of epiboly (gastrulation), 12 somites (neurulation) and 24hpf stage (organogenesis). Altogether, the goal of this project is to understand how developmental signaling pathways control CREs activity and regulate gene expression over different stages during the embryonic development.

Trabajo presentado en el EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021.

Trabajo presentado en el EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021.

Trabajo presentado en EMBO Workshop Enhanceropathies: Understanding enhancer function to understand human disease, celebrado en Santander (España) del 06 al 09 de octubre de 2021.

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021., Retinoic acid (RA) is the active form of retinol (vitamin A) and functions as a ligand for the nuclear RA receptors (RARs), which regulate the expression of target genes by binding to RA response elements (RAREs). RA signaling is required for embryonic development in chordate animals, being involved in multiple processes, such as body axis extension, hindbrain antero-posterior (AP) patterning, differentiation of neuro-mesodermal progenitors, heart AP patterning and forelimb bud initiation, among others. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during embryonic development. Here we use ChIP-seq to profile the genomic binding of RAR in zebrafish embryos at different developmental stages. Stimulating the RA pathway by treating embryos with all-trans-RA (atRA), the most abundant form of RA, we use a combination of RNA-seq, ATAC-seq and ChIP-seq to gain insight into the mechanisms by which RA signaling control the expression of direct and indirect target genes during early development. Finally, we explore the interplay between RA signaling, Hox and Meis transcription factors, which have been proposed to cooperate during axial skeleton AP patterning. Our results provide new clues about the crosstalk between signaling pathways and transcription factors during vertebrate embryonic development.

Trabajo presentado en EMBO Workshop The evolution of animal genomes, celebrado en modalidad virtual del 13 al 17 de septiembre de 2021.

© The Author(s) 2020., How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development., The work was supported by Wellcome grants 088566, 097820 and 105610, with additional support from MRC project grants MR/L009986/1 to N.B. and N.A.H., MR/ J003352/1 to K.P.H., and MR/000638/1 and MR/S036121/1 to N.A.H. R.E.J. was an MRC clinical research training fellow, and S.J.W. was an MRC doctoral account PhD student. J. L.G.S. was supported by the Marató TV3 Fundacion (Grant No. 201611).

Trabajo presentado en el VII Congress of the Spanish Society for Evolutionary Biology / VII Congreso de la Sociedad Española de Biología Evolutiva (SESBE VII), celebrado en Sevilla del 5 al 7 de febrero de 2020., Genes undergoing substantial evolutionary shifts in their expression profiles are often modulated by critical epigenomic changes that are among the primary targets of selection in evolution. Here, we investigate the evolution of epigenetic regulatory activities and their interplay with gene expression in human and non-human primate lineages. We extensively profiled a new panel of human and non-human primate lymphoblastoid cell lines using a variety of NGS techniques and integrated genome-wide chromatin contact maps to define gene regulatory architectures. We observe that epigenetic and sequence conservation are coupled in regulatory elements and reflect the impact of their activity on gene expression. The addition or removal of strong and poised promoters and intragenic enhancers is frequent in gene expression changes during recent primate evolution. In contrast, novel human-specific weak intragenic enhancers, dormant in our cell lines, have emerged in genes showing signals of recent adaptive selection, suggesting that they echo important regulatory innovations in other cell types. Among the genes targeted by these regulatory innovations, we find key candidate drivers of recently evolved human traits, such as FOXP2 or ROBO1 for speech and language acquisition, and PALMD for neocortex expansion, thus highlighting the importance of regulatory changes in human evolution.

Trabajo presentado en Belgian Society of Human Genetics meeting “The Epigenome in Development and Disease”, celebrado en Gante (Bélgica) el 16 de febrero de 2018.

17th Euro Fed Lipid Congress 20-23 October 2019, Seville, Spain, Histone modifications play an integral role in plant development. Investigating chromatin organization in developing seeds and its role in regulating gene expression allows us to understand the mechanisms underlying fatty acid biosynthesis during oil accumulation process and identify novel functional regions in oleaginous plant genomes. Oleaginous seeds pose a challenge for using high-throughput chromatin immunoprecipitation (ChIP) techniques due to the rich oils could seriously affect inmunprecipitation and DNA isolation. In this study we investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene expression during different stages of the seed development using ChiP-seq in sunflower (Helianthus annuus). Profiles of annotated genes involved in fatty acid biosynthesis associated to chromatin regions upregulated or downregulated have been analyzed. Identification of sequences associated to the master regulator of the fatty acid biosynthesis WRI1 has been also carried out.

Trabajo presentado en Brown Bag Lunch Seminars (2018), celebrado en Chicago (Estados Unidos) el 24 de julio de 2018.

Resumen del póster presentado en el 7th meeting of the European Society for Evolutionary Developmental Biology (EED), celebrado en Galway (Irlanda) del 26 al 29 de junio de 2018., All chordates share a fundamental bodyplan that was greatly elaborated in vertebrates. Vertebrates also evolved highly distinctive genomes, sculpted by two whole genome duplications (WGD) and the acquisition of unique genomic traits. These traits include numerous features often associated with gene regulation, such as unusually large intergenic and intronic regions and a distinct set of highly conserved non-coding regions. To investigate the evolution of these features and genome regulation in chordates, we characterized promoters, methylation, chromatin accessibility, histone modifications and transcriptomes in multiple tissues and throughout development of the cephalochordate amphioxus, a slow-evolving nonvertebrate chordate. These data revealed high conservation of gene expression and its underlying cis-regulatory logic between amphioxus and vertebrates, maximally at a developmental phylotypic period. We also found a strong increase in the complexity of vertebrate gene regulatory landscapes, specially among genes retained in several copies after the two vertebrate WGDs. Finally, we unraveled the principal route of regulatory evolution following WGD: over 80% of gene families with multiple paralogs in vertebrates have members that restricted their ancestral expression, undergoing specialization rather than subfunctionalization. Counter-intuitively, vertebrate genes that underwent expression restriction increased the complexity of their regulatory landscapes. Altogether, these data pave the way for a better understanding of the regulatory principles underlying key vertebrate innovations.

Trabajo presentado en Decision making in cell collectives: mechanisms in development, homeostasis and evolution launching meeting, celebrado en Carmona (Sevilla) del 05 al 06 de abril de 2018.

Trabajo presentado en Society for Molecular Biology and Evolution (SMBE), celebrado en Yokohama (Japón) del 08 al 12 de julio de 2018.

Resumen del póster presentado al 1st Joint Meeting of the French-Portuguese-Spanish Biochemical and Molecular Biology Societies y al XL Spanish Society of Biochemistry and Molecular Biology (SEBBM) Congress, celebrado en Barcelona (España) del 23 al 26 de octubre de 2017., p63 transcription factor plays an important role during epidermis and neural crest derivatives formation. Mutations affecting its coding sequence or its target genes lead to the development of malformations mainly characterized by cleft palate, ectodermal dysplasia and limb malformation. To date, the search of genetic events underlying these malformations has been mainly focused on exonic regions. However, impairment on the transcriptional regulation of genes related to these phenotypes might cause similar diseases. The major problem of the studies focused on transcription factors binding sites is the difficulty to associate a certain regulatory region with its target gene. In general, this association is solely established having into account the proximity between these elements, which might lead to wrong assignments and waste of effort and resources in deeply studying the incorrect genes. To overcome this problem, we designed an approach based on binding sites and chromatin 3D interactions, which will allow us to properly associate transcriptional regulatory elements with their target genes. First, we performed a ChIP-seq in zebrafish to find p63 binding sites, and then 4C-seq experiments centered in some of these putative regulatory regions to uncover their regulatory landscape. Finally, we moved to genome-wide scale by means of a novel technique, the HiChIP, which allowed us to obtain a global map of contacts from every active promoter and assign the most probable target gene to each binding site. The integration of these data will provide us a list of genes contacting p63-binding regions, which could potentially belong to the p63 genetic network and be involved in p63-related diseases.

Resumen del trabajo presentado a la 50º Reunión Anual de la Sociedad de Genética de Chile y a la XI Reunión Anual de la Sociedad Chilena de Evolución, celebradas en Puerto Varas (Chile) del 9 al 11 de noviembre de 2017., A pesar de su importancia desde el punto de vista evolutivo, del desarrollo y funcional, el origen de los apéndices pareados de vertebrados sigue siendo un misterio. Las ideas clásicas se centran en tres hipótesis: que las aletas apareadas se relacionan con los arcos branquiales, que derivan de un pliegue lateral, o que las aletas medias evolucionaron primero y las aletas pareadas surgieron por co-opción de antiguos módulos genéticos presentes en las aletas dorsales. Combinando ensayos transgénicos en pez cebra y ratón, estudios del paisaje epigenético de Shh en diferentes animales y ensayos de CRISPR/Cas9 en peces, aquí demostramos no solo la historia evolutiva de un elemento cis-regulador esencial para la función de Shh en apéndices, sino también su función ancestral que nos revela el origen evolutivo de nuestras extremidades.

Resumen del póster presentado al Joint Congress of the Spanish Societies of Genetics, Cell Biology and Developmental Biology, celebrado en Gijón del 24 al 27 de octubre de 2017.-- et al., During embryonic development, the behaviour of thousand of cells must be tightly coordinated by a precise cellular crosstalk. Despite this complexity, communication between cells is orchestrated by only a few signalling pathways highly conserved across animals. In response to these pathways, different cis-regulatory elements are activated and repressed, controlling, in a spatio-temporal manner, the expression of their target genes. Although these signalling pathways have been extensively studied, our knowledge of the cis-regulatory changes associated with their activation or repression is still limited. Here, we aim to explore this issue using Danio rerio as vertebrate model. In our laboratory, we are currently manipulating different signalling pathways using agonist and antagonist molecules to further profile open chromatin regions in both treated and control embryos. Using this strategy, we have recently identified a retinoic acid (RA) responding enhancer in intron eight of the zebrafish meis2b gene. This gene encodes a TALE homeobox protein with multiple functions during vertebrate development. Sequence analysis of this enhancer reveals a strong binding site for the RA receptor, suggesting a direct control of meis2b by this signalling pathway. Using zebrafish transgenic assays, we have determined that this enhancer promotes expression in the hindbrain, a RA-dependent territory that shows strong meis2b transcription. The intron-exon organization of the Meis genes is extremely conserved during evolution. Indeed, we have found RA-responding enhancers in the same intron of most meis paralogous in zebrafish. We have also validated with 4C-seq data that some of these enhancers contact the promoter of the meis genes. Interestingly, we have also detected open chromatin regions in the equivalent introns in the amphioxus, sea urchin and flies Meis orthologous. We are currently using zebrafish enhancer assays to examine if the introns from these species contain functionally evolutionary conserved enhancers, and to determine the evolutionary origin of the Meis gene expression control by the RA signalling pathway.

Resumen del trabajo presentado al Joint Congress of the Spanish Societies of Genetics, Cell Biology and Developmental Biology, celebrado en Gijón del 24 al 27 de octubre de 2017., Animal development requires the precise transciptional regulation of thousands of genes in every cell of the embryo. Interestingly, transcriptional regulation in metazoans seems to be heavily influenced by the 3D organization of the genome in several ways. On the one side, active and inactive chromatin regions tend to cluster together in the nucleus forming the so called A and B compartments. Some developmental genes are known to switch from one compartment to another while getting activated or repressed. On the other side, developmental genes are often controlled by large sets of enhancers that activate them in a spatio-temporal specific manner during embryogenesis. In order to regulate the transcription from a promoter, enhancers need to be close to them. Specific 3D structures named chromatin loops can facilitate the interaction in space between enhancers and their target promoters, even though they might lay far apart in the linear sequence. In the same way, undesired interactions between enhancers and promoters can also be prevented at the structural level. Chromatin conformation capture techniques, such as HiC, have been key to understand these roles of the 3D configuration of the genome in gene regulation. In short, HiC experiments provide the frequency of interactions between every two pair of loci in the genome. It has been shown recently that by coupling the regular HiC protocol with a step of chromatin selection with a given antibody it is possible to sample only those interactions involving a protein of interest, such as CTCF or cohesin. In this work, we perform HiChIP experiments in 24hpf zebrafish embryos using antibodies against three histone marks: H3K4me3 (marking active promoters), H3K27ac (marking active promoters and enhancers), and H3K27me3 (marking polycomb repression) reaching 5kb resolution with a moderate sequencing effort. We provide evidence that the HiChIP experiments using H3K4me3 and H3K27ac antibodies are sufficient to predict active promoters and enhancers at 24 hpf stage and assign these predicted enhancers to their targets promoters, all at once. In addition, HiChIP using the H3K27me3 antibody confirmed that the presence of polycomb mediated repressive interactions, such as those taking place between the HoxD and Dlx1-2 gene clusters in mouse, are conserved in zebrafish. Compartments A and B are also readily identified using this approach.

Ponencia presentada en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en la Universidad Jaume I (Castellón, España) del 23 al 25 de septiembre de 2015., Dorso-ventral pigment pattern differences are the most widespread pigmentary adaptations in vertebrates. In mammals, agouti-signaling protein (ASIP) have been reported as the key factor in this process by regulating melanin types and levels in melanocytes. In fish, studies of pigment patterning have focused on stripe formation, identifying a core striping mechanism dependent upon interactions between different pigment cell types. We have recently demonstrated thatzebrafish utilize two distinct adult pigment patterning mechanisms -an ancient dorso-ventral patterning mechanism, and a more recent striping mechanism based on cell-cell interactions; remarkably, the dorso-ventral patterning mechanism also utilizes Asip. We hypothesize that Asip have a conserved role in the countershading pigment pattern formation in vertebrates. However, the molecular and cellular mechanisms underlying this dorso-ventral asip1 expression gradient remain largely unexplored. We report here the asip expression in the holostei spotted gar (Lepisosteus oculatus), one of Darwin's defining examples of 'living fossils'. Using phylogenetic footprinting and transgenesis in zebrafish, we examine the conserved cis-regulatory elements surrounding agouti gene., This work was funded by Spanish AGL2011-23581 project.

Resumen del trabajo presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.-- et al., Compartmentalization of cells during development is a fundamental process that allows the segregation of precursor populations into distinct tissue domains. The vertebrate hindbrain (HB) is subdivided along the antero-posterior axis in 7 rhombomeres (Rs) emerging during development through a finely tuned cascade of TFs that confer identity to each R. In zebrafish, each rhombomere is flanked by actomyosin cables that prevent cells to intermingle. These cables form physical boundaries that keep neuroblasts confined to their precise location within the HB. Here we found that the small GTPase Rac3b is expressed at the HB boundaries and regulate actomyosin cables assembly. In addition, Rac3b was found to be part of a synexpression group of genes only found in teleost fish from the Ostariophysi superorder, a group that comprise 68% of fresh water species. Comparative genomics and chromosomal conformation capture analysis suggest that after the teleost WGD, the novel expression pattern emerged in the Ostariophysi lineage by a chromosomal inversion causing the fusion of two highly conserved TADs. Using an enhancer reporter vector we found two CREs that drive expression to R boundaries. A full deletion (Δ2,5kb) of both enhancers was generated using the CRISPR/Cas9 system. Phenotypic analysis of this mutant points to the existence of additional CREs (i.e. shadow enhancers) within the novel TAD directing the expression of these genes to the boundaries. The observed redundancy stresses the relevance of this novel mechanism for rhombomere segregation during development.

Comunicación presentada en The International Symposium on Genetics in Aquaculture XII, celebrado en Santiago de Compostela, España, del 21 al 27 de junio de 2015, Pigment abnormalities are one of the leading causes in performance loss in at sh aquaculture. Nevertheless, and in spite of the importance of the problem, there is scanty information about the molecular regulation of sh pigmentation which makes the control of this problem di cult. To date, the most important regulator of skin and hair pigmentation in vertebrates is the Agouti signaling protein (ASIP)- melanocortin 1 receptor (MC1R) system: the agouti locus encodes the Agouti-signaling protein (ASIP), which antagonizes the e ects of -Melanocyte- Stimulating hormone (-MSH) binding to the MC1R on the follicular melanocyte, resulting in a decrease in the production of eumelanins (dark pigments) and an increase in pheomelanins (red/yellow pigments). A key aspect of mammalian pigment pattern, that is widely conserved, is the pale ventrum and darker dorsum; the underlying mechanism is regulation of ASIP expression, with high ventral Agouti driving pale belly color. Likewise, sh often show a pale ventrum, but here the pigmentation mechanism is very di erent with independent cell-types, each expressing chemically distinct pigments (not just melanins), being di erentially distributed. Recently, we have shown that not only the agouti gene (asip1), but also even the elevated ventral expression levels, are conserved in sh. It is of interest, in order to know the molecular regulation of sh pigmentation, to establish how Asip1 functions to regulate sh pigment pattern formation. In order to explore the mechanism of Asip1 in zebra sh pigment pat tern formation, we used a BAC transgenic approach to allow characterization of asip1 cis-regulation regions. We have generated a BAC transgenic zebra sh line in which 5 and 3 agouti locus regulatory regions direct eGFP overexpression. Our initial observations showed a graded dorso-ventral expression of eGFP. Therefore, eGFP expression in the asip1- iTol2-eGFP-BAC transgenic sh faithfully recapitulated the endogenous expression pattern of asip1 mRNA. This opens the way to characterizing the mechanisms generating this crucial graded distribution of asip1, This work was funded by the Spanish Science and Innovation Ministry project ALG2011-23581 to JR. Laura Cal was supported by PhD fellowship FPI BES-2012-055414.

The development of organs composed by repeated functional units is, in many cases, accomplished by the transition of cells from a progenitor to a differentiation domain, triggering a reiterated developmental program. Yet, how these discrete fields are formed during development is still a largely unresolved question. The posterior lateral line (pLL), a sensory organ present in fish and amphibians, develops from a primordium that migrates along the flanks of the animal periodically depositing neuromasts, the pLL functional units. In zebrafish (Danio rerio), the developmental program of the pLL is triggered by the transit of progenitor cells from a Wnt to a Fgf signaling domain. It has been proposed that these two fields are defined by the antagonistic activity of these two signaling pathways, but how they are formed and maintained is still an open question in the development of the pLL. In this work, we show that sox21a, an HMG -box transcription factor, is expressed within the Fgf domain. We demonstrate that, while the Fgf signaling pathway do not control sox21a, knockdown of sox21a causes impairment of Fgf signaling, expansion of the Wnt signaling domain and disruption of neuromast development. These results suggest that sox21a is a key player in the pLL primordium patterning, fine-tuning the border of the Fgf and Wnt signaling domains., Funded by: Spanish and Andalusian Governments Grant Numbers: BFU2010-14839, CSD2007-00008, and Proyecto de Excelencia, CVI-3488 to JLG-S; FEDER (Operational Competitiveness Programme—COMPETE); National Funds (FCT—Fundação para a Ciência e a Tecnologia project FCOMP-01-0124-FEDER-041232, (EXPL/BEX-BID/0713/2013)]; CSIC (FPI fellow to A.A.); ERC Reseal (A.B.B.) . Grant Number: 2007-StG-208631; FCT investigator (J.B. was a Juan de la Cierva postdoctoral fellow of the CSIC) Grant Number: IF/00654/2013.

Comunicación presentada en el 10º Congreso de la Asociación Ibérica de Endocrinología Comparada AIEC, celebrado en Castellón, España, del 23 al 25 de septiembre de 2015

Under a Creative Commons license.-- et al., A rare germline duplication upstream of the bone morphogenetic protein antagonist GREM1 causes aMendelian-dominant predisposition to colorectal cancer (CRC). The underlying disease mechanism is strong, ectopic GREM1 overexpression in the intestinal epithelium. Here, we confirm that a common GREM1 polymorphism, rs16969681, is also associated with CRC susceptibility, conferring ~20% differential risk in the general population. We hypothesized the underlying cause to be moderate differences inGREM1 expression. We showed that rs16969681 lies in a region of active chromatin with allele- and tissue-specific enhancer activity. The CRC high-risk allele was associated with stronger gene expression, and higher Grem1 mRNA levels increased the intestinal tumor burden in ApcMin mice. The intestine-specific transcription factor CDX2 and Wnt effector TCF7L2 bound near rs16969681, with significantly higher affinity for the risk allele, and CDX2 overexpression in CDX2/GREM1-negative cells caused re-expression of GREM1. rs16969681 influences CRC risk through effects on Wnt-driven GREM1 expression in colorectal tumors. © 2014 The Authors., Funding was provided from Cancer Research UK grant A/16459, an EU FP7 SYSCOL Consortium grant, and the EU COST colorectal cancer initiative. Core funding to the Wellcome Trust Centre for Human Genetics was provided from the Wellcome Trust (090532/Z/09/Z). J.L.G.-S. and J.J.C. were supported by the Spanish/FEDER government grants BFU2010-14839 and BFU2011-2292., Open Access funded by Wellcome Trust.

Resumen del trabajo presentado al Symposium Reading and Interpreting the Epigenome: Challenges and Opportunities, celebrado en la University of Western Australia el 18 de diciembre de 2014., The generation of distinctive cell types that form different tissues and organs requires precise, temporal and spatial control of gene expression. This depends on a vast amount of highly dynamic cis-regulatory elements distributed in the non-coding DNA. To address how this cis-regulatory information is organized in the vertebrate genome, we have employed 4C-seq to determine the regulatory landscape of multiple genes in zebra fish and mouse embryos. Our studies demonstrate that most of these genes display striking developmental and evolutionarily conserved 3D architecture that is likely essential for their correct regulation during evolution and development. Investigation of regulatory landscapes of a number of these genes in the cephalochordate amphioxus and the sea urchin genome has revealed that some of these 3D architectures have an evolutionary ancestral origin whereas others are vertebrate innovations. An enhanced understanding of 3D regulatory landscapes during development and evolution also allows us to point out unexpected genes associated with human diseases. Through our 4C-seq methodology we have recently linked alterations in 3D chromatin structure to the diabetes-associated SNPs.

The locomotory appendages of vertebrates have undergone significant changes during evolution, which likely promoted a wide range of adaptive strategies. These appendages first evolved as unpaired finfolds in the dorsal midline of early chordates, more than 500 million years ago. Later on, during vertebrates' radiation, two sets of locomotory appendages emerged, developing from both sides of the latero-ventral body wall. The morphology of these paired fins in fishes at different phylogenetic positions suggests an evolutionary tendency for increasing elaboration of the endoskeleton and concomitant reduction of the distal dermoskeleton. This evolutionary process culminated with the origin of limbs in the lineages leading to tetrapods. The developmental programs responsible for the evolution of vertebrate appendages have been a major topic for evolutionary developmental biology recently. Gene expression comparisons performed in chordates explored how these mechanisms were transferred from a midline to latero-ventral position. On another front, gene function assays have begun to test classical hypotheses concerning the transition from fish fins to tetrapod limbs. In this review, we highlight these recent findings on the evolution of vertebrate fin development. First, we discuss new perspectives on the transition from midline to paired appendages focus on (i) origin and molecular regionalization of the lateral plate mesoderm and (ii) novel ectodermic competency zones for fin induction. Next, we review recent work exploring how tetrapod limbs evolved from fish fins, considering (i) molecular and structural changes in the distal ectoderm of fins and (ii) modulation of 5′HoxD transcription during fin endoskeleton development., Portuguese Foundation for Science and Technology; grant number: EXPL/BEX‐BID/0801/2013

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License., The origin and evolution of the complex regulatory landscapes of some vertebrate developmental genes, often spanning hundreds of Kbp and including neighboring genes, remain poorly understood. The Sonic Hedgehog (Shh) genomic regulatory block (GRB) is one of the best functionally characterized examples, with several discrete enhancers reported within its introns, vast upstream gene-free region and neighboring genes (Lmbr1 and Rnf32). To investigate the origin and evolution of this GRB, we sequenced and characterized the Hedgehog (Hh) loci from three invertebrate chordate amphioxus species, which share several early expression domains with Shh. Using phylogenetic footprinting within and between chordate lineages, and reporter assays in zebrafish probing >30 Kbp of amphioxus Hh, we report large sequence and functional divergence between both groups. In addition, we show that the linkage of Shh to Lmbr1 and Rnf32, necessary for the unique gnatostomate-specific Shh limb expression, is a vertebrate novelty occurred between the two whole-genome duplications., M.I., D.B., I.M. and J.G.-F. were funded by Grants BFU2005-00252 and BMC2008-03776 and BMC2011-23291 from the Spanish Ministry of Science and Innovation, and J.G.F. by the ICREA Academia Prize. M.I., D.B. and I.M. held FPI, APIF-UB and FPU fellowships, respectively. J.-L.G.-S. and J.L.R. were supported by Grants BFU2010-14839, CSD2007-00008875 (MEC), and CVI 3488 (Junta de Andalucía). JLR holds a JAE-Doc grant from the National Research Council, founded by Social European Funds.

Resumen del póster presentado a la 1ª Jornada de los másteres de biotecnología de la UPO, celebrada en Sevilla el 3 de mayo de 2012., Six3 and Six2 are transcription factors from the SIX family of homeoproteins. The Six genes play important roles during development controlling patterning, cell proliferation and migration. Across bilaterian evolution, Six3 and Six2 genes have been maintained in very close proximity in the genome despite displaying largely different expression patterns. This suggests that these genes are exposed to different regulatory environments. Using zebrafish as a model, we are trying to precisely define the regulatory landscape for each gene and determine how and why synteny is maintained and differential expression patterns is generated. For this goal, we use our zebrafish histone epigenomic data (Rada-Iglesias, 2011) to select and test putative enhancers (Bessa et al., 2009) for both genes. We also use chromatin conformation capture (3C) derived technologies (Splinter et al., 2011) to elucidate the global chromatin architecture of each promoter. In addition, BAC recombineering will allow us to test the influence of regulatory elements and potential insulators on gene expression and chromatin architecture of the locus. In this presentation I will talk about the theoretical context of the project, methodological set up and work in progress.

Resumen del trabajo presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.

Resumen del trabajo presentado al 6th Annual NCMLS Symposium: New Frontiers in Personal Genomic, celebrado en Nijmegen (Holanda) del 3 al 4 de diciembre de 2012., Only 5-10% of vertebrate genomes consist of coding sequences or genes. Moreover, the vast majority of these genes are highly conserved in all species. How then did morphological diversity arise during evolution? The current genetic theory of morphological evolution states that, to a great extent, evolution of form is the history of changing gene expression during development. Gene expression depends on cisr-egulatory regions thata control where, when and how much a gene is transcribe. These regulatory regions, immersed in the vast sea of non-coding DNA, need also to be organized in defined tridimensional chromatin architectures to precisely control their target genes. Gene regulations is not only crucial for development, but is also essential for controlling the physiology of cellsin adult organisms. Therefore, mutations that affect the activity cis-regulatory elements or alter the tridimensional chromatin architecture may not only affect development or contribute to evolution, but are associated to multiple human disease. Accordingly, many of the large number of genome-wide association studies that have been reported in the last few years indicate that the lesions associated with human genetic diseases are usually not in the coding DNA of candidate genes, but rather in the non-coding regulatory regions associated with them. In the cases, the tissue, the timing or the amount of mRNA produced, and therefore of protein, is abnormal causing human disorders. This lecture will discuss the importance of a tridimensional chromatin structure for the correct regulation of the Iroquois genes, a family of homeoproteins encoding genes that play critical functions during animal development and are associated in gene clusters. We will also demonstrate how alteration in this chromatin structure is linked to human diseases.

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.-- et al.

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.

Resumen del póster presentado al IX Meeting of the Spanish Society for Developmental Biology celebrado en Granada del 12 al 14 de noviembre de 2012.

Resumen del póster presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre del 2011., El factor de transcripción inducible por hipoxia (HIF) juega un papel fundamental en la respuesta transcripcional a hipoxia, y por tanto en la adaptación a bajas concentraciones de oxígeno. En el presente trabajo hemos tratado de profundizar en las bases de la selectividad de los genes dianas de HIF, utilizando como paradigma la respuesta transcripcional a hipoxia de una pareja de genes: GYS1 y RUVBL2, de transcripción opuesta pero con sus inicios de transcripción a menos de 1kb. A pesar de que el elemento de respuesta a hipoxia (HRE) se localiza entre ambos genes, únicamente los niveles de mRNA de GYS1 incrementan en respuesta a hipoxia, mientras que los niveles de mRNA de RUVBL2 permanecen constantes. Sin embargo, la falta de inducción de RUVBL2 no se debe a su silenciamiento, dado que en un amplio panel de líneas celulares los niveles de mRNA en condiciones de normoxia son similares o incluso mayores que los de GYS1, al igual que la actividad promotora de las construcciones indicadoras en normoxia. Utilizando diferentes construcciones derivadas de este locus, hemos determinado que la región localizada entre el HRE y el promotor de RUVBL2 previene la inducción de este gen en condiciones de bajo oxígeno, ya que su eliminación permite la inducción de su actividad promotora en respuesta a la activación de HIF. Ensayos de bloqueo de enhancer in vitro también parecen indicar la existencia de un aislador genómico en ésta región. Además, el promotor de RUVBL2 presenta actividad aisladora en ensayos de bloqueo de enhancer desarrollados in vivo en Zebrafish. En este trabajo se describe por primera vez un aislador genómico que actúa frente a un estímulo ambiental como es la hipoxia y explica cómo se consigue la selectividad de HIF sobre sus dianas.

Resumen del póster presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre del 2011., Los genomas de vertebrados están organizados funcional y estructuralmente en dominios, que incluyen todos los elementos reguladores necesarios para la correcta expresión de los genes que contienen. Estos dominios están delimitados por aisladores genómicos, elementos reguladores con propiedades de bloqueo de enhancers y de barrera, y que contribuyen a la organización estructural del locus mediante la interacción con la arquitectura nuclear. Se han descrito multitud de secuencias de unión a CTCF, un factor nuclear asociado a aisladores genómicos en vertebrados, utilizando métodos de análisis masivo. Sin embargo, nuestras publicaciones recientes demuestran que existen secuencias aisladoras que no requieren CTCF (por ejemplo, SINE). Por ello hemos desarrollado nuevos algoritmos bioinformáticos, para el análisis sistemático del genoma humano y de ratón, utilizando herramientas como aGEM, que permiten inspeccionar el patrón de expresión de genes en diversos tejidos y compararlos frente al de sus vecinos inmediatos, centrándonos en aquellos que presenten diferencias y explorando las secuencias intergénicas correspondientes, en particular aquellas que hayan sido conservadas evolutivamente. Para validar funcionalmente estos aisladores genómicos aplicamos el método diseñado en el laboratorio. Primero, realizamos un test in vitro de bloqueo de enhancer en células de mamífero en cultivo. En segundo lugar, evaluamos estos mismos elementos in vivo, usando construcciones similares en pez cebra. Los aisladores descritos con este método pueden funcionar en construcciones heterólogas, como transgenes y vectores de terapia génica, combatiendo eficazmente los efectos de posición cromosomales.

9 páginas, 7 figuras., The extracellular signal-regulated kinase (ERK) is a key transducer of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) signaling pathways, and its function is required in multiple processes during animal development. The activity of ERK depends on the phosphorylation state of conserved threonine and tyrosine residues, and this state is regulated by different kinases and phosphatases. A family of phosphatases with specificity toward both threonine and tyrosine residues in ERK (dual-specificity phosphatases) play a conserved role in its dephosphorylation and consequent inactivation. Here, we characterize the function of the dual-specificity phosphatase MKP3 in Drosophila EGFR and Xenopus FGFR signaling. The function of MKP3 is required during Drosophila wing vein formation and Xenopus anteroposterior neural patterning. We find that the expression of the MKP3 gene is localized in places of high EGFR and FGFR signaling. Furthermore, this restricted expression depends on ERK function both in Drosophila and Xenopus, suggesting that MKP3 constitutes a conserved negative feedback loop on the activity of the Ras/ERK signaling pathway., Funded by Ministry of Science and Technology. Grant Numbers: BCM2000-1191, GEN2001-4846-C0501, BMC2001-2122. Dirección General de Investigación Cientófica y Técnica. Fundación Ramón Areces.

The Iroquois (Iro) family of genes are found in nematodes, insects and vertebrates. They usually occur in one or two genomic clusters of three genes each and encode transcriptional controllers that posses a characteristic homeodomain. The Iro genes function early in development to specify the identity of diverse territories of the body, such as the dorsal head and dorsal mesothorax of Drosophila and the neural plate of Xenopus. In some aspects they act in the same way as classical selector genes, but they display specific properties that place them into a category of their own. Later in development in both Drosophila and vertebrates, the Iro genes function again to subdivide those territories into smaller domains. Grants from Human Frontier Science Program (RG0042/98B), Comunidad Autónoma de Madrid (08.5/0044.1/99) and Dirección General de Investigación Científica y Técnica (PB98-0682), and an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa are acknowledged.

Programa de Doctorado en Biotecnología e Ingeniería Bioquímica, Retinoic Acid (RA), a lipophilic molecule anda metabolite of Vitamin-A (all-trans-Retinol),is a well known morphogen that affects gene lranscription and modulales a wide variety of biological processes in vertebrales. The largets of all-trans-Retinoic Acid include a multilude of structural genes,oncogenes, transcription factors and cytokines.In this work we use a unique collection of zebrafish reporter lines generated in our l ab lo detect tissue specific downstream targels of RA. Here we present preliminary results suggesting lhat mycb, an oncogene whose misregulation has been relatad to different types of cancers, is repressed specifically in the branchial arches by RA, situation that leads to a decrease in cell proliferation. In addition, we are analyzing the genomic landscape of this gene using Circularized Chromosome Conformation Capture (4C). By this way we will identify genome wide DNA sequences that interact with the promoter of mycb which might correspond to elements of lhe regulatory landscape of this gene. Another proyect in course is dealing wilh pancreatic cell differentiation. We are analyzing elements of the Salvador/Warts/Hippo pathway that seems lo be active during zebrafish pancreas development. The Hippo tumor suppressor palhway restricts organ size in Drosophila and mammals by antagonizing the oncoprotein Yki/YAP. Previous Chip-seq experiments for pancrealic transcription faclors suggest that the downstream targets of Hippo signaling pathway tead and yap could be importan!for a proper expression of developmental genes in pancreas. In this work we show preliminary results from the manipulation of the Hippo signaling pathway suggesting that it is active also in zebralish and that yap and tead play a crucial role in pancreas development by controlling cell proliferation and differenilation., Universidad Pablo de Olavide. Centro de Estudios de Postgrado

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología Química Línea de Investigación: Bioinformatica en Biotecnología y Biomedicina Clave Programa: DBI Código Línea: 7 Animal morphological diversity is astonishing and it is partially due to differences in gene expres- sion between different species during development. Recently, the genome folding in Topologically Associated Domains (TADs) found in most animals has been shown to be critical in the control of transcription during development. Distal enhancers are able to interact with the promoters of developmental genes only when they belong to the same 3D environment or TAD. Therefore, we investigated how changes in the 3D folding of the genome could have impacted the changes in gene regulation responsible for the evolution of vertebrates. In order to do so we combined syntenic analysis with Chromatin Conformation Capture experiments such as 4C-seq and HiChIP. First we compared the chromatin folding around the zebrafish HoxD and the amphioxus Hox loci using 4C-seq experiments coupled to computational modelling. The chromatin architecture around the vertebrate HoxD locus is peculiar, with al the HoxD genes located at the boundary between two TADs allowing them to switch to respond to distal enhancers located in either of the two TADs during the patterning of the limbs. In contrast, all the amphioxus Hox genes belong to the same TAD. However, the region located downstream from Hox1 is homologous to the vertebrate anterior TAD and is wired both in 3D and functionally to the regulation of Hox genes also in amphioxus. This suggests a stepwise evolution of the chromatin folding in two TADs found in extant vertebrates, with the anterior TAD being already wired to Hox genes in the last common ancestor of chordates. Second we performed a genome wide comparison of the chromatin folding between zebrafish and amphioxus using HiChIP and antibodies against different histone modifications. Using H3K4me3 HiChIP experiments we were able to identify the Regulatory Landscapes (RLs) of all active devel- opmental promoters using a single experiment. By doing so we were able to identify almost four hundred cases of chromosomal rearrangements that potentially altered the boundaries of a TAD and were susceptible to generate regulatory novelties in the vertebrate lineage. Also, we found that the two events of whole genome duplication that occurred at the root of vertebrates allowed some of the paralog genes originated to increase their RLs both in size and in number of enhancers. Universidad Pablo de Olavide de Sevilla. Centro de Estudios de Posgrado Postprint

Programa de Doctorado en Biotecnología y Tecnología Química, Estudio de los mecanismos de evolución y desarrollo de vertebrados: Análisis del impacto de los cambios en transen el patrón de expresión de los genes y estudio del papel de sox21a en la linea lateral. Durante la evolución de vertebrados los organismos han adquirido grandes diferencias fenotípicas,las cuales se pueden explicar por cambios en el patrón de expresión de los genes. Por tanto, es muy importante conocer como se regula la expresión génica. Parte de la regulación génica es debida a la existencia de secuencias no codificantes activadoras de la transcripción, conocidas como secuencias reguladoras en cis. Es interesante, conocer como se modifica la actividad de estas secuencias durante la evolución. Existen dos posibilidades para cambiar la actividad de estas secuencias reguladoras, o bien modificando la secuencia propiamente dicha (evolución en cis) o bien modificando la maquinaria transcripcional que lo regula (evolución en trans). A lo largo de esta tesis hemos intentado responder a como los cambios trans impactan en el patrón de expresión de los genes, durante la evolución de vertebrados. Para responder a esta pregunta hicimos ensayos de actividad cis reguladora en pez cebra y las comparamos con el patrón de expresión generado por la misma secuencia en ratón. Con los resultados obtenidos introducimos un nuevo objetivo en éste trabajo de doctorado. Hacer un estudio de potenciales secuencias cis-reguladoras específicas de teleósteos. Para ello nos hemos centrado en el gen sox21a el cual tiene un dominio de expresión específico en el pez cebra, la línea lateral. La línea lateral es un órgano mecano-sensorial presente en peces y anfibios. Durante el desarrollo, ésta estructura es formada por un grupo de células que migra desde la vesícula ática hasta el final de la cola durante este viaje se van depositando las estructuras funcionales de la línea lateral, los neuromastos. Dos morfógenos juegan un papel muy importante en el desarrollo de la línea lateral. Wnt es requerido para mantener el estado proliferativo de las células y FGF que desencadena los pasos de diferenciación y epiteliarización. Sin embargo, se desconocen quienes son los reguladores de estos dos morfogenos. Diferentes genes de la familia Sox se expresan en la línea lateral entre ellos se encuentra sox21a. Estudios en sistema nervioso han demostrado que este gen es requerido para la transición de células proliferativas a diferenciadas.Intentando responder a cual era el papel de sox21a en la linea lateral; usando morfolino de sox21a y haciendo estudios in vivo y observamos que en la condición morfante se producía un retraso en la migración, una falta en la epiteliarización, así como una inhibición de los genes expresado en el dominio Fgf. Estos resultados sugieren que sox21a es un regulador positivo que actúa aguas arriba de Fgf. Por tanto, postulamos que sox21a juega un papel muy importante en el establecimiento de las diferentes identidades celulares en la línea lateral., Universidad Pablo de Olavide. Centro de Estudios de Postgrado

Tese de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto por Ana Maria Bastos Barros Neto e realizado em el Centro Andaluz de Biología del Desarrollo, centro mixto CSIC-UPO., [EN]: Odd-skipped family of proteins (Odd in Drosophila and Osr in vertebrates) encode evolutionary conserved zinc-finger (ZF) transcription factors. In mammalians two oddskipped related (osr) genes were found, Osr1 and Osr2. At the moment we start this study the function of the vertebrate proteins was completely unknown. In the first part of this PhD project, we characterize the expression pattern of osr1 and osr2 genes in Xenopus and in zebrafish. We reported the role of osr genes in the development of renal structures (pronephros) in vertebrates. We found that osr1 and osr2 are both necessary and sufficient for the development of pronephros. Molecularly, osr proteins function as transcriptional repressors in renal development. We also showed that Drosophila odd induces the formation of kidney tissue in Xenopus. This ability is dependent of the Engrailed homology1 (eh1) domain. Odd genes may also be required for proper development of the Malpighian tubules, the Drosophila renal organs. Our results highlight the evolutionary conserved involvement of Odd-skipped transcription factors in the development of kidneys. In the second part of this PhD project we set up to study the involvement of these genes in pectoral fin development, unravelling their place in the genetic cascade. Osr genes are expressed in the pectoral fin domain around 48hpf onwards. We found, by loss of function experiments, that osr genes are required for the formation of the pectoral fin bud. Strikingly, effect of loss of function is traced back to the 21 somites stage, affecting the expression of the earliest fin/limb specification marker, tbx5. We hypothesized that osr, expressed in the intermediate mesoderm, might act indirectly in the lateral plate mesoderm. We found that osr1 and both osr genes are able to regulate wnt2ba expression, a diffusible molecule, with a described role in fin development. Moreover, RA signalling is able to modulate osr genes expression, this controlling kidney and fin formation. Taken together, these results bring up a novel function of osr genes in the early steps of fin development. In collaboration with Rankin and colleagues, we revealed the requirement of osr genes in the development of some endoderm structures such as lung and liver. The involvement of osr genes in the formation of endoderm derivatives as well as the molecular signalling, using wnt2b, seem to be conserved in Xenopus and zebrafish., [PT]: A família de proteínas “Odd-skipped” é denominada como Odd na mosca da fruta (Drosophila) e em vertebrados por Osr, e são dedos de zinco altamente conservados durante a evolução. Em mamíferos dois genes “odd-skipped related” (osr) foram descobertos, Osr1 and Osr2. No princípio deste estudo a função das proteínas osr era totalmente desconhecida. Na primeira parte deste projecto de doutoramento, o padrão de expressão dos genes osr1 e osr2 foram caracterizados em Xenopus e no peixe zebra. Neste estudo, a função destes genes foi descrita no desenvolvimento de estruturas renais (pronefros) em vertebrados. Podemos concluir que ambos genes, osr1 e osr2, são necessários e suficientes para o desenvolvimento de pronefros. As proteínas osr actuam a nível molecular como repressores da transcrição durante o desenvolvimento renal. Também demonstrámos que a proteina odd de Drosophila tem a propriedade de induzir a formação de tecido renal em Xenopus. Esta capacidade é dependente do domínio “Engrailed homology1“ (eh1). Os genes Odd podem tambem ser necessários para o correcto desenvolvimento dos tubulos de Malpighi, os orgãos renais da Drosophila. Os resultados apresentados neste trabalho ressaltam a conservação a nivel evolutivo dos factores de transcrição “Odd-skipped” no desenvolvimento renal. Na segunda parte deste projecto de doutoramento proposemos o estudo da função destes genes no desenvolvimento das barbatanas peitorais, clarificando o lugar destes genes na via de sinalização. Os genes osr genes são expressos no domínio das barbatanas peitorais a partir de cerca de 48 horas depois da fertilização. Neste estudo demonstrámos por experiências de perda de função que os genes osr são necessários para a formação do primórdio das barbatanas peitorais. O efeito da perda de função é detectado no estadio de 21 somitos, afectando a expressão do marcador mais precoce de especificação da barbatana/membro, tbx5. Neste contexto os genes osr s ão expressos na mesoderme intermédia e poderiam actuar indirectamente na placa de mesoderme lateral. Neste trabalho descrevemos que osr1 e ambos genes osr regulam a expressão de wnt2ba, que é uma molécula difusível e com uma função já descrita no desenvolvimento das barbatanas. A sinalização do ácido retinóico é capaz de regular a expressão dos genes osr, e consequentemente a formação dos rins e das barbatanas. Em conjunto estes resultados demonstram a importância dos genes osr nas primeiras etapas de desenvolvimento das barbatanas. Em colaboração com Scott Rankin e o grupo de Aaron Zorn, estudámos a importância dos genes osr no desenvolvimento de estruturas derivadas da endoderme como o pulmão e o fígado. A função dos genes osr na formação de derivados da endoderme assim como a sinalização molecular, via wnt2b, parece estar conservada em Xenopus e no peixe zebra., Este trabalho foi financiado pela Fundação para a Ciência e Tecnologia.